Oxidative metabolism in smooth muscle cells: Methods and materials relating thereto

ABSTRACT

The application concerns oxidative metabolism in smooth muscle cells and to methods and materials related thereto. Provided is a method which is a medium suitable for the support of oxidative metabolism comprises: 1) incubating a cell or tissue sample with a specimen derived from a patient wherein the sample comprises smooth muscle cells; 2) measuring a marker of oxidative metabolism of the cell or tissue sample; and 3) detecting an increase in oxidative metabolism which is not attributable to a contractile demand for ATP, such as an increase in oxidative metabolism being indicative of the existence in a patient of an agent or combination of agents able to cause an increase in oxidative metabolism in smooth muscle cells.

This application is a divisional of U.S. Ser. No. 09/174,581, filed Oct.19, 1998, now U.S. Pat. No. 6,080,385, which is a continuation ofPCT/GB97/01086, filed Apr. 18, 1997.

The present invention relates to oxidative metabolism in smooth musclecells and to methods and materials relating thereto.

In particular, the present invention relates to methods and materialsrelating, for example, to

(i) diagnosis of the existence or onset of chronicvasospasm/vasoconstriction

(ii) monitoring treatment of chronic vasospasm/vasoconstriction

(iii) identifying new drugs effective in the treatment of chronicvasospasm/vasoconstriction

(iv) the elucidation of agents (“spasminogens”) causing chronicvasospasm/vasoconstriction.

Vasospasm/vasoconstriction represents a significantly preventable causeof morbidity and death. Vascular smooth muscle (VSM) is able to maintaintension for extended periods at low energy cost (the phenomena is knownas “latch”). This is essential for the autonomous and continuousregulation of blood flow to the organs etc. However invasospasm/vasoconstriction, there is an abnormal contraction of theblood vessels to a vascular bed combined with the blood vessels having adiminished ability to relax. This restricts the blood flow and inconsequence the oxygen supply. A variety of vascular beds including egcardiac, mesenteric, placental, uterine and cerebral may be affectedwith consequent serious clinical implications such as organ damage,stroke, death or miscarriage (Rajani, R. M., B. V. Dalvi, S. A. D'Silva,Y. Y. Lokhandwali and P. A. Kale (1991) Postgrad. Medical Journal 67(783) 78-80 and Gewertz B. L. and C. K. Zarins (1991) J. Vasc. Surg. 14382-385.)

The term “vasospasm” is generally used in relation to contraction of theblood vessels particularly associated with the brain. In contrast, theterm “vasoconstriction” is generally used in relation to constrictionsof the blood vessels associated with organs other than the brain.Hereafter, where the term vasospasm is used it should not be interpretedsolely as a reference to a vascular spasm associated with the brainunless the context otherwise demands such interpretation.

The invention of the present application is disclosed with reference toa number of clinical conditions linked with abnormal metabolismassociated with vasospasm/vasoconstriction, namely: cerebral vasospasmin consequence of sub-arachnoid haemorrhage; pre-eclampsia andAlzheimer's disease. Conditions other than those specifically named willbe known to those skilled in the art as being associated with chronicspasm/constriction of smooth muscle cells, such as chronicvasospasm/vasoconstriction.

Cerebral vasospasm occurs as a result of a sub-arachnoid haemorrhage.The haemorrhage strikes without warning, mostly in young adults andaffects about 16 in every 100,000 people each year. The conditionaccounts for about 10% of all cerebrovascular disease.

The arachnoid layer lies between the pia meter (which wraps the brainand closely follows its contours) and the outermost dural layer. Itcomprises two arachnoid membranes separated by the sub-arachnoid spacewhich is a cerebral spinal fluid (CSF) filled cavity containing bloodvessels which supply oxygen to the brain. Thus, these blood vesselssupplying the brain are bathed in CSF. A sub-arachnoid haemorrhageoccurs when a blood vessel in the sub-arachnoid space ruptures.Typically the rupture results from an aneurysm. Blood leaks from thevessel into the sub-arachnoid space where it mixes with the CSF.

In 30% of sub-arachnoid haemorrhage patients, the rupture to the bloodvessel results in death before the patient reaches the hospital. Theremaining 70% survive the initial damage to the blood vessel and areadmitted to hospital. In these admitted patients, the damaged vesselconstricts to prevent excess blood loss and blood flow is reduced. Thisinitial constriction in the acute phase of the condition is known as avasoconstriction and is a part of the normal vessel repair responseprocess. With the constriction, the repair of the rupture commences withthe formation of a clot. In some patients (about 60% of those admitted)the vessel eventually re-dilates following occlusion of the rupture andblood flow returns to normal with consequent good clinical outcome andreturn to good health. Unfortunately, and despite considerable clinicalefforts, other patients (the remaining 40% of those admitted)deteriorate. The deterioration may be the result of a vessel re-bleed orhydrocephalus (excess CSF in the cranial vault) and in suchcircumstances the patient can be treated by surgery and stands arelatively good chance of survival. Alternatively, the deterioration maybe due to the delayed onset of prolonged and irreversible vasospasm(hereafter referred to chronic vasospasm). Unless the condition isdiagnosed prior to its onset and effectively treated by drug therapy orsurgery, it can lead to stroke, brain oedema or death.

In order to prevent the onset of chronic vasospasm in sub-arachnoidhaemorrhage patients, one needs to be able to accurately diagnose thosepatients who are likely to undergo the condition. The presentlyavailable diagnostic methods do not allow one to predict the likelyonset of chronic vasospasm in sub-arachnoid haemorrhage patients. It isonly possible to diagnose the actual presence of chronic vasospasm andthis is determined by performing an angiogram in order to determine thecause of the bleed and also to look for evidence of vascularconstrictions.

Of the 70% of patients who survive the initial bleed and undergo anangiogram, 60% show evidence of vasospasm. Only 40% of these willactually develop neurological deterioration. However, some patients candevelop a delayed neurological deterioration without evidence ofangiographic vasospasm, but with reduced blood flow. Therefore, in thisgroup of patients the data suggests that an angiogram is not an accuratemethod of diagnosing the onset of chronic vasospasm.

There is also a time window for performing successful surgery, as ifsurgery is performed between 3 and 14 days after sub-arachnoidhaemorrhage, the risk of chronic vasospasm actually increases. Theresults from angiograms are not sufficiently predictive of the optimaltime for undertaking surgery.

Thus there is a need for a method which allows one to predict as soon aspossible those patients likely to go into chronic vasospasm, such thatappropriate and/or required intervention can be initiated within thefirst three days following the cerebral haemorrhage.

In addition to the absence of suitable diagnostic methods, there is notat present an effective treatment for chronic cerebral vasospasm. Todate, the condition is treated by angioplasty to the narrowed artery andby use of the Ca²⁺ antagonist, Nimodipine (Nimotop*, Bayer). However,results have shown that although treatment with Nimodipine produces asmall (20%), but significant improvement in clinical outcome forpatients, it does not alter the vessel lumen size and therefore does notreverse the constrictive effects of the cerebral vasospasm (Pickard, J.D., G. D. Murray and R. Illingworth (1989) Brit. Med. Journal 298636-642).

Indeed it is possible that the use of calcium antagonists may becounterproductive. The prior art to date indicates that cerebralvasospasm is the result of metabolic failure of the VSM cells and thatbreakdown products of red blood cells within CSF can alter the energymetabolism of VSM as evidenced by decreases in phosphocreatine and ATP(Kim, P., J. Jones and T. M. Sundt (1992) J. Neurosurg. 76 991-996) andan increase in ADP. ADP has been shown to inhibit VSM cross-bridges(Clark J. F., Z. Khuchua, A. V. Kuznetsov, A. E. Boehm and R.Ventura-Clapier (1994) J. Musc. Res. Cell motil. 15 432-439 and Clark J.F., G. J. Kemp and G. K. Radda (1995) J. Theor. Biol. 173 207-211) andtherefore elevated ADP may lead to an inability of vascular smoothmuscle (VSM) to relax. This suggestion fits with the observation that aCa²⁺ antagonist such as (Nimodipine) cannot change the contractilestate, as ADP maintained tension is independent of Ca²⁺ (Clark et al.,(1994) supra). Therefore the use of anything which results in anelevation of ADP is undesirable. A Ca²⁺ antagonist can decreasecytosolic Ca²⁺. The decrease in cytosolic calcium may lead to anelevation of ADP because of decreased mitochondrial function due to lackof Ca²⁺ stimulation.

Glucocorticoid steroids, antioxidants, anti-prostaglandin agents,endothelin antagonists and other vaso-dilators have all failed toprovide any clinical benefits despite initially encouraging animalstudies.

Thus there remains a real need for agents effective in the treatment ofclinical conditions associated with chronic vasospasm/vasoconstriction,such as cerebral vasospasm following sub-arachnoid haemorrhage.

Investigations into the pathological significance of the metabolicfailure in canine basilar arteries after sub-arachnoid haemorrhageshowed that the ATP, GTP and PCr content of the arteries diminishedrapidly over the 14 days post-haemorrhage, with significant decreasesbeing seen from day 3 (Kim et al., (1992) supra). The same investigatorsin a study looking at the high energy phosphate levels in the caninecerebral artery during chronic vasospasm, found that the levels of ATP,GTP and PCr were also decreased in these spastic arteries and that theratios of ATP:ADP, GTP:GDP and PCr:Cr were decreased. Further, totaladenine and creatine contents were diminished.

This association between metabolic failure and irreversible VSM cellcontraction is supported by observations that: (i) a decrease in ATPleads to a rigor-like status in VSM cells; (ii) exposure of VSM cells to250 μM intracellular ADP results in an inability of the cells to relaxproperly (Clark et al., (1994) supra); and (iii) application ofintracellular ADP to contracted VSM cells impairs their ability to relaxin a dose dependant manner (Nishye, E., A. V. Somlyo, K. Torok and A. P.Somlyo et al., (1993) J. Physiol. 460 247-271).

Thus, the thinking based on the prior art studies to date, is thatmitochondria are deficient in the production of ATP, thus decreasing theATP:ADP ratio and causing a relative increase in cellular ADP levels,there being a failure of the oxidative metabolism. The increased levelsin cellular ADP lead to prolonged contraction of the smooth muscle cellsand to their inability to relax. The reduction in high energy phosphateswas measured in correlation with vasospasm, and indicated that the onsetof chronic vasospasm is correlated to a deficiency in high energyphosphates.

However, contrary to the above teachings, the present inventors havemade the unexpected and surprising discovery that increased ADP levelsin VSM cells following sub-arachnoid haemorrhage is in consequence of anincrease in flux through the oxidative metabolism pathway, rather thandue to mitochondrial deficiency.

When VSM cells are maximally stimulated under working conditions,oxidative metabolism is double that of the basal rate observed innon-working conditions. Uncoupling mitochondria with an agent such asdinitrophenol causes them to consume oxygen without producing ATP. Themaximum rate of oxygen consumption observed in uncoupled mitochondria is3 times the basal rate.

The present inventors have made the unexpected discovery that if oneapplies eg CSF supernatant from the sub-arachnoid space of a patient whohas suffered a sub-arachnoid haemorrhage, CSF supernatant fromAlzheimer's disease patients or sera from pre-eclampsia patients, to aportion (eg. a strip or ring) of porcine carotid artery (which comprisesVSM cells) under non-load conditions such that any increase in oxygenconsumption observed cannot be due to contraction of the strip (no loadmeans no contractile demand for ATP), there is a significant increase(typically a 6 time increase) in the rate of oxygen consumption ascompared to basal rate consumption along with an increase in ATPase. Theincrease in rate of oxygen consumption develops over several hours, butis clearly evident after only a 1 hour delay and maintained for 5 ormore hours.

This discovery is contrary to the teachings of the prior art asdiscussed above i.e. rather than the respiratory system failing/dying,there is an increase in flux. If, as the prior art teaches, the observedincrease in ADP is in consequence of metabolic failure, one would notexpect to see an increase in cellular oxygen consumption.

Thus the evidence of the present inventors suggests that in a clinicalcondition associated with chronic spasm/constriction of smooth musclecells (eg chronic vasospasm/vasoconstriction as in cerebral vasospasm inconsequence of sub-arachnoid haemorrhage, pre-eclampsia and Alzheimer'sdisease), there is a spasminogen (this term being used to refer to anagent or combination of agents which cause the increase in oxidativemetabolism in smooth muscle cells (such as vascular smooth muscle cellsleading to chronic vasospasm/vasoconstriction) not attributable to acontractile demand for ATP) present in body fluids such as CSF or blood(in cerebral haemorrhage, blood mixes with CSF in the sub-arachnoidspace), which appears to stimulate ATPase eg actinomyosin ATPase insmooth muscle such as VSM and is the likely initiator of chronicspasm/constriction. The mitochondria respond to the increased ATPaseactivity by consuming increased amounts of oxygen to produce ATP, butare unable to maintain metabolic homeostasis and thus ADP levelsincrease. The increased levels of ADP appear to be the cause ofirreversible spasm/constriction of the blood vessels.

In relation to cerebral vasospasm, research into the nature of thespasminogen currently falls into two groups: (a) indirect vasoactivecompounds such as inflammatory compounds (prostaglandins, thromboxanes,leukotrienes, and blood breakdown products); and (b) direct vasoactivecompounds such as Ca²⁺ ions, angiotensin and endothelin.

Current theory suggests that in cerebral vasospasm the spasminogen(s) isreleased into the CSF from the blood, where effects causing constrictionof VSM cells are mediated. Endothelin 1 which has been implicated incoronary artery vasospasm (Igarashi K., M. Horimota and T. Takenaka,(1993) J. Cardiol. 23 257-262; Davies M. G., M. L. Klyachkin, J. H. Kimand P. O. Hagen (1993) J. Cardiovasc. Pharmacol. 22 Suppl. 8 S348-S351;Chester A. H., G. S. O'Neil, S. P. Allen and T. N. Luu (1992) Eur, J.Clin. Invest. 22 210-213) and mesenteric artery vasospasm (Yoshida M.,a. Suzuki and T. Itoh (1994) J. Phsiol. Lond. 477 253-265) has beenproposed as a likely spasminogen although there is also evidence thatinduction of vasospasm may be mediated by a multitude of compounds(Toyo-oka T., T. Aizawa, N. Suzuki, Y. Hirata, T. Miyauchi, W. S. Chin,M. Yanagisawa, T. Masaki and T. Sugimoto (1991) Circulation 83 476-483).

Turning now to pre-eclampsia and Alzheimer's disease.

Pre-eclampsia is the foremost cause of maternal death and iatrogenicprematurity in the UK. It predisposes to intrauterine growthretardation, foetal asphyxia and foetal death. The features of thematernal syndrome are remarkably diverse including hypertension,proteinuria, abnormal clotting and multiple organ dysfunction. Cerebralpathology associated with the syndrome comprises one of the commonestcauses of maternal death. Eclampsia, characterised by epileptiformconvulsions, is thought to arise from such intense vasoconstriction asto cause focal cerebral ischaemia, even infarction. The origins of thepathology are obscure, but the associated cerebral vasospasm isconfirmed by magnetic resonance angiography (Matsuda et al., 1995Gynecol. Obstet. Invest. 40, 249-252) and computed tomographic imaging.Pathological vasoconstriction which occurs during cerebral vasospasm, isalso a component of the hypertension and other problems of pre-eclampsiaincluding, apart from eclampsia itself, the liver dysfunction and foetaldistress, which are all characteristic of the syndrome. The diversefeatures of the syndrome results in the diagnosis of the condition beingvery subjective and in consequence inherently unreliable. There is aneed for more reliable methods which allow one to predict either thelikelihood of the condition occurring or the presence of the condition.

Alzheimer's disease is a major cause of memory loss and dementia inelderly people. Depositing of two types of abnormal filaments arecharacteristic brain lesions seen in Alzheimer's patients: the mainlyintraneuronal neurofibrillary tangles which consist of paired helicalfilaments and the extracellular amyloid fibers and phosphorylation ofτ(tau) protein. To date, a firm diagnosis of Alzheimer's disease is madeon the basis of the presence of large numbers of these abnormalfilamentous structures in the brain.

However the diagnosis can only be carried out in conjunction with majorsurgery of an invasive nature. Generally this diagnosis is carried outpost-mortem. Therefore there is a real need for alternative/additionaldiagnostic methods which are less invasive.

The discussion above makes it clear that improved diagnostic andtreatment methods are needed in relation to certain medical conditionssuch as chronic cerebral vasospasm, pre-eclampsia and Alzheimer'sDisease which are related to the presence of an agent or combination ofagents able to cause an increase in oxidative metabolism in smoothmuscle cells such as VSM cells and hence an abnormal metabolic stateassociated with chronic spasm/constriction (egvasospasm/vasoconstriction).

The present invention provides a new way of both predicting a patient'slikelihood of suffering a pathological condition which is in consequenceof the presence of an agent or combination of agents able to cause anincrease in oxidative metabolism in smooth muscle cells (eg VSM cells)and hence an abnormal metabolic state associated with spasm of smoothmuscle cells (such as chronic cerebral vasospasm following fromsub-arachnoid haemorrhage; pre-eclampsia, Alzheimer's Disease) anddiagnosing a patient as having such a pathological condition and/orabnormal metabolic state. The present invention also provides ways of(i) identifying spasminogens present in biological samples which arecritically involved in inducing chronic spasm/constriction; (ii)identifying new drugs for preventing or ameliorating suchspasms/constrictions; (iii) monitoring the effectiveness of anytreatment administered to a patient to either prevent or ameliorate suchspasms/constrictions.

The diagnostic or predictive method comprises:

in a medium suitable for the support of oxidative metabolism incubatinga cell or tissue sample with a fluid specimen deriving from a patient,wherein the sample comprises smooth muscle cells;

measuring a marker of oxidative metabolism of the cell or tissue sampleand detecting an increase in oxidative metabolism which is notattributable to a contractile demand for ATP, such an increase beingindicative of the existence in the patient an agent or combination ofagents able to cause an increase in oxidative metabolism in smoothmuscle cells not attributable to a contractile demand for ATP.

Where there is in a patient an agent or combination of agents able tocause an increase in oxidative metabolism in smooth muscle cells notattributable to a contractile demand for ATP, the increase in oxidativemetabolism may be unusual/abnormal and of clinical significance.

The increase in oxidative metabolism may be significant and sustained.Thus the increase in oxidative metabolism may be of a level representedby an increase in oxygen consumption of about three times or greater.The increase may be represented by an increase in oxygen consumption ofapproximately five times increase. The increase in oxidative metabolismmay be sustainable for greater than one hour, usually several hours, forexample for 5 or more hours.

The method can be used to diagnose a patient as having, or predictingthat a patient will in future have an abnormal metabolic state (whichimpacts on the contractile state as metabolism through ADP productionwill alter the contractile state of smooth muscle cells) characterisedby an increase in oxidative metabolism in smooth muscle cells notattributable to a contractile demand for ATP. The sample may comprisevascular smooth muscle cells and the abnormal metabolic state may beassociated with chronic vasospasm/vasoconstriction and clinicalconditions related thereto. Although species and vascular bed cross-overis perfectly possible and exemplified herein, the vascular smooth musclecells may be representative of those involved in the suspected existingor predicted vasospasm/vasoconstriction of a vascular bed. The methodmay be employed in relation to the diagnosis, prognosis, predictionand/or quantification of a medical condition such as pre-eclampsia,Alzheimer's Disease or chronic cerebral vasospasm.

In relation to chronic cerebral vasospasm, if the specimen causes anincrease in oxidative metabolism that is not attributable to acontractile demand for ATP (such a specimen may be described as “hot”)and is obtained from a patient within the first three dayspost-haemorrhage this indicates that the patient is at high risk ofgoing into vasospasm. However under such circumstances one wouldgenerally re-test eg daily or twice daily within the few weekspost-haemorrhage to monitor changes in the likelihood of developingvasospasm. Risk of vasospasm may increase or recede.

Conversely, if the specimen has no effect on oxidative metabolism (sucha specimen may be described as “cold”) this indicates that the patientis not at high risk of going into vasospasm. However, under suchcircumstances one may wish to regularly re-test the patient (eg. dailyor twice daily) within the two weeks post-haemorrhage to monitor changesin the likelihood of developing vasospasm.

The test results will influence the medical or surgical treatment.Generally speaking, where the specimen proves to be “cold” surgery islikely to be safer.

Thus where the specimen is “cold” one may operate irrespective of thetiming of surgery with respect to the initial haemorrhage (traditionallyone would delay for 14 days before operating, but such delay may beunnecessary where the specimen is cold).

Where the specimen is “hot” the options are to delay operating untilsuch time as a “cold” specimen is obtained, or proceed with surgery inthe knowledge that there is an increase in risk of the patient goinginto vasospasm and that the patient will need to go into an intensivecare unit for close observation and treatment.

Generally, where one is wanting to identify a spasminogen (ie an agentor combination of agents able to cause an increase in oxidativemetabolism in smooth muscle cells such as VSM cells, not attributable toa contractile demand for ATP) one may be aided by a method whichinvolves

in a medium suitable for the support of oxidative metabolism incubatinga cell or tissue sample which comprises smooth muscle cells with aspecimen thought to contain a said spasminogen and

measuring a marker of oxidative metabolism of the cell or tissue sample;and

detecting an increase in oxidative metabolism which is not attributableto a contractile demand for ATP, such an increase in oxidativemetabolism being indicative of the presence of said spasminogen in thespecimen.

The smooth muscle cells may be vascular smooth muscle cells. Thevascular smooth muscle cells may be of a type representative of thosethought to be affected in vivo by the spasminogen.

The biological specimen may comprise a fraction of a specimen as derivedfrom a patient. Fractions may be derived in accordance with knownmetabolite or protein purification techniques such as phase separation,electrophoresis, chromatography and mass spectrometry. The method maycomprise the additional step of purifying the spasminogen from thebiological sample in accordance with protein purification techniques andsmall molecule purification techniques known in the art.

Thus the invention provides any new spasminogen (an agent or combinationof agents able to cause an increase in oxidative metabolism in smoothmuscle cells (eg vascular smooth muscle cells) not attributable to acontractile demand for ATP) readily identifiable by use of the methodsas herein described.

Spasminogens or patient derived specimens (eg sera) identified as beingable to cause an increase in oxidative metabolism in smooth muscle cellseg vascular smooth muscle cells, not attributable to a contractiledemand for ATP can be used in the methods herein described to screen forcompounds useful as drugs to counter or block the effects of thespasminogen.

Generally where one is wanting to screen compounds for potentialefficacy as therapeutic agents the method involves:

in a medium suitable for the support of oxidative metabolism

incubating a cell or tissue sample which comprises smooth muscle cellswith (a) a specimen, agent or combination of agents known to effect anincrease in oxidative metabolism of the cell of tissue sample which isnot attributable to a contractile demand for ATP; and (b) a testcompound;

measuring a marker of oxidative metabolism; and

detecting an absence or reduction of the increase in oxidativemetabolism not attributable to a contractile demand for ATP and which isnormally effected by the specimen, agent or combination of agents, anysuch absence or reduction being indicative of the test compound havingpotential efficacy as a therapeutic agent.

The cell or tissue sample may comprise vascular smooth muscle cells.

The cell or tissue sample can be incubated with the test compound priorto incubation with the specimen, agent or combination of agents.Alternatively, the cell or tissue sample can be incubated with the testcompound and specimen, agent or combination of agents substantiallysimultaneously. In either of these approaches, the absence or reductionof the increase in oxidative metabolism which is not attributable to acontractile demand for ATP, can be detected by comparison to a controltest, which is identical except for being without the test compound.

In the alternative, in a medium suitable for the support of oxidativemetabolism the cell or tissue sample can be incubated with the specimen,agent or combination of agents prior to incubation with the testcompound and for a time sufficient to establish an increase in oxidativemetabolism which is not attributable to a contractile demand for ATP.The test compound is then added and one then can measure the marker ofoxidative metabolism in order to detect a reduction of the increase inoxidative metabolism which is not attributable to a contractile demandfor ATP normally associated with the specimen, agent or combination ofagents.

The present invention therefore also provides new compounds andtreatment methods identified following use of an approach as hereindescribed and the use of such drugs in the preparation of medicamentsfor the prevention or treatment of abnormal metabolic or contractilestates characterised by an increase in oxidative metabolism in smoothmuscle cells (eg vascular smooth muscle cells) not attributable to acontractile demand for ATP. The prevention/treatment may be of a chronicvasospasm/vasoconstriction. The prevention/treatment may be in relationto pre-eclampsia, Alzheimer's Disease or chronic cerebral vasospasm.

Where one is wanting to monitor the effectiveness of any treatmentadministered to a patient to either prevent or ameliorate an abnormalmetabolic state or chronic spasm/constriction (eg chronicvasospasm/vasoconstriction) or symptoms of pre-eclampsia, Alzheimer'sDisease or chronic cerebral vasospasm, the method involves carrying outa method of diagnosis as earlier described and repeating the method atone or more appropriately selected time intervals to detect analteration of the increase in oxidative metabolism which is notattributable to a contractile demand for ATP and wherein a reduction ofthe increase is indicative of the treatment being therapeuticallyadvantageous.

The incubations can be carried out in any medium suitable for thesupport of oxidative metabolism. One suitable incubation medium is Krebsbuffer with 11 mM glucose and/or 5 mM pyruvate. Another isKrebs-Hensleit buffer containing 0.5 mM KH₂PO₄ with either 11 mM glucoseor 5 mM pyruvate as the substrate. The incubations may be aerated with95%O₂-5%CO₂. The pH may be 7.4. Other mediums and incubation conditionssuitable for the methods described herein are known and available tothose skilled in the art.

The cell or tissue sample used may be one which is representative ofthose involved in the spasm. For example, if the spasm is of vascularsmooth muscle, the sample should comprise VSM cells and therefore thetissue sample may comprise a suitable culture of VSM cells or a strip oftissue from eg basilar, carotid, coronary or mesenteric arteries ofpigs, guinea pigs or humans, or rat aorta. If one is concerned withmeasuring spasm associated with a particular organ, one may select thecell culture or tissue accordingly, eg for chronic cerebral vasospasmone may select carotid arteries or basilar arteries and for cardiacvasospasm one may select coronary artery or aorta. In relation topre-eclampsia one may use cells or tissue samples deriving from theuterine or placental vascular beds or from the umbilical artery.

If one is concerned with spasm of non-vascular smooth muscle cells, thesample should be selected accordingly and the cell culture/tissues mayderive from eg intestine endometrium, myocardium, or skeletal muscletissue. Cell culture samples may be used eg A7RS VSM cells derived fromembryonic rat aorta (ECACC, PHLS Centre for Applied Microbiology andResearch, Salisbury, Wiltshire, United Kingdom SP4 0JG).

Generally speaking, the specimen may be fluid specimen such as anyderivable from a patient (eg. CSF, blood, urine, sera plasma, pentonealfluid, pericardial fluid, pleural fluid) and which is likely to containthe spasminogen if present. Obviously, the choice of fluid specimen foranalysis will be dictated by the nature of the patient's condition, butin many cases the fluid sample will comprise blood or serum. For chroniccerebral vasospasm resulting from sub-arachnoid haemorrhage, the fluidspecimen may comprise CSF which has mixed with blood in thesub-arachnoid space in consequence of the haemorrhage.

The marker of oxidative metabolism may be oxygen consumption rate,ATPase activity (see example 6 and Clark, J. F. and Dillon, P. F., 1995J. Vasc. Res. 32, 24-30), kinase activity ADP concentration (Fisher, M.J., and Dillon, P. F., 1988 NMR in Biomed. 1, 121-126), all of whichwill increase in line with an increase in oxidative metabolism. Analternative marker of oxidative metabolism is the phosphorylation stateof myosin (Dillon, P. F. et al., 1981 Science 211, 495-497) and othersuitable markers will be apparent to those skilled in the art.Techniques for measurement of various markers indicating the state ofoxidative metabolism are known to those skilled in the art. For example,where one chooses to measure oxygen consumption rate as a marker ofoxidative metabolism, the incubation may be carried out in an air-tightvessel incorporating an oxygen sensor for measurement of the oxygenconcentration of the incubation solution. Alternatively, one may measuremarkers such as ATPase activity in the cell or tissue sample, ADPconcentration in the model cell or tissue, phosphorylation state ofmyosin or other contractile proteins associated with myosin in the cellor tissue all in accordance with known techniques and as referred to inthe papers stated above.

The incubation period may be any period sufficient to allow detection ofan increase in oxidative metabolism. The examples provided herein givethose skilled in the art some guidance, but the skilled person would beable to determine an appropriate incubation period. Generally, where thespecimen is peripheral blood and the tissue is vascular smooth muscle inthe form of a strip of porcine carotid artery an incubation period inexcess of 120 mins may be necessary. Where one is concerned with chroniccerebral vasospasm and the specimen is CSF, the incubation period may beshorter than this. Other detailed or chronic analysis may require aslong as 2 days. Of course as stated earlier alternatives to the specificform of tissue eg vascular smooth muscle tissue, used to exemplify thediscoveries and invention may be employed eg smooth muscle cells fromcell culture.

In the above, one is looking for any increase in oxidative metabolismwhich is not attributable to a contractile demand for ATP. Thus thesmooth muscle cells of the sample may be without a contractile demandfor ATP ie they are not contracting. Where the smooth muscle cells ofthe model cell or tissue sample are without a contractile demand for ATPany increase in oxidative metabolism is indicative of a spasminogenbeing present in the specimen.

An increase in oxidative metabolism above 3 times basal rate should betaken as strongly indicative of a spasminogen being present in thespecimen.

In the alternative, the smooth muscle cells of the sample may have acontractile demand for ATP. In which case, one looks for an increase inoxidative metabolism which is not attributable to that contractiledemand. This may be done by comparison to a control incubation in whichthe cells have a contractile demand for ATP, prior to application of thefluid specimen. One may then compare any increases in oxidativemetabolism before and after addition of the fluid sample. As analternative, a test incubation may be compared to a ‘separate’ controlincubation; ‘separate’ in that it is a different incubation comprising aseparate, but comparable model cell or tissue sample, treated/incubatedidentically except for the addition of the fluid sample.

The present invention also provides a method of treating a patient withsub-arachnoid haemorrhage and at risk of going into chronic vascularvasospasm which comprises administering to the patient: (a) a compoundwhich puts VSM cells into the contracted state and/or (b) a compoundwhich blocks the effects of the spasminogen.

Histamine is an example of a compound which puts VSM cells into thedephosphorylated state following contraction (known as the “latched”state). Vascular smooth muscle contraction requires myosin light chainkinase to phosphorylate Myosin ATPase, and myosin ATPase to interactwith actin before it can generate tension. It is unique however, whencompared to other muscles because it can maintain tension for extendedperiods with low energy costs (Dillon, P. F. et al 1981 Science 211,495-497; Lynch, R. M. and Paul, R. J. 1987 Am.J.Physiol. 256, c328-c334;Hai, C. M. and Murphy, R. A. 1988 Am.J.Physiol. 254, c99-c106).Increased intracellular calcium (Ca⁺⁺) concentration, which stimulatesmyosin light chain kinase, is needed to generate but not to maintaintension (Dillon, P. F. et al., 1981 supra). Indeed, near maximal tensioncan be maintained with Ca⁺⁺ concentrations below those necessary to halfmaximally activate myosin light chain kinase (Hai, C. M. and Murphy, R.A. 1988). This condition is achieved by de-phosphorylating myosinATPase, when attached to actin, effectively ‘locking’ the two proteinstogether. This ‘locked together’ state is called latch and isinsensitive to Ca⁺⁺ stimulation (Hai, C. M. and Murphy, R. A. 1988).Numerous other enzymes however, are sensitive to cytosolic Ca⁺⁺ such asKrebs cycle enzymes (Drummond, R. M. J. V. et al., 1995 Biophysics J.68, A230) and its is though that calcium activation of the mitochondriamay be essential during periods of high metabolic flux.

Putting VSM cells into the “latched” state appears to prevent VSMexperiencing an increase in respiratory flux in consequence of contactwith the spasminogen.

Dobutamine which also puts VSM cells into the “latched” state, appearsto block the effects of the spasminogen.

Therefore the present invention provides treatment methods based on bothhistamine and dobutamine.

A suitable treatment may comprise use of 500 mg dobutamine in 50 mlsnormal saline. Infusion commencing to give 1 mg/hour rising to 10-15mg/hour. Infusion may be continued for a period (generally 24-48 hours)sufficient to give the desired blood pressure response (eg ¹²⁰/₉₀) orrenal output response.

The present invention also provides use of compounds as set out in (a)and (b) above the histamine and dobutamine, in the preparation ofmedicaments for the treatment of chronic cerebral vasospasm.

The present invention provides an assembly of a test apparatus and acell or tissue sample wherein:

the test apparatus comprises a vessel, a lid to render said vesselair-tight and an oxygen sensor for measurement of the oxygenconcentration of any solution in said vessel; and

the cell or tissue sample comprises a portion human basilar artery or asample of vascular smooth muscle cells.

The assembly may comprise a stirring means within the vessel. Theassembly may comprise a liquid medium for location in the vessel.Suitable mediums are hereafter described.

The incubation mediums used in the methods and assemblages describedabove may be free of any material comprising cells which may beundergoing metabolic processes. Thus they may be free of viablemicroorganism such as bacteria. The mediums may be rendered free ofviable microorganisms by use of suitable sterilising techniques, filters(eg filters for removal of bacteria) or other methods known to thoseskilled in the art.

In using a method as stated above to identify a spasminogen whichinduces spasm of smooth muscle cells, the present inventors have madethe unexpected discovery that okadaic acid stimulates an increase inoxidative metabolism in porcine carotid artery. The effect caused byokadaic acid is very similar to that caused by subarachnoid haemorrhagesupernatant. In investigating this further, the inventors have obtainedexperimental results which indicate that the physical properties of thesubstance in subarachnoid haemorrhage supernatant which acts as aspasminogen appear to be consistent with those of okadaic acid.Specifically both are acid stable, they both have hydrophilic andhydrophobic domains, they both have a molecular weight of less than 1000daltons. Okadaic acid has a molecular weight of 805 daltons, it islipophilic, protein philic (but not a protein itself).

Okadaic acid is a lipophilic compound which is a phosphatase-specificinhibitor. It is known to cause smooth muscle constriction. It wasoriginally found in molluscs. Inhibition of the de-phosphorylation ofmyosin ATPase would cause an increased ATPase activity (see earlier inthis text).

The present inventors therefore suggest that: (i) the pathophysiologicalmechanism for chronic vasospasm/vasoconstriction is likely to involvephosphatase inhibition or loss of kinase and/or ATPase control; and (ii)the spasminogen is likely to comprise a molecular entity which bringsabout phosphatase inhibition. Alternatively the spasminogen may be amolecular entity which brings about activation of a protein kinase. Theresult of inhibition of protein phosphatase activity is an increase inthe population of phosphorylated proteins. This is also the result whenprotein kinases (eg protein kinase C “PKC”) are activated. Indeed,phorbol esters such as phorbol-meristate will cause a significantactivation of PKC in vascular smooth muscle (and other tissues). Invessels, PKC activation results in the phosphorylation of Calponin andCaldesmon. These proteins are inhibitory proteins of myosin ATPase andwhen phosphorylated their inhibition of myosin ATPase is removed.Therefore, by active phosphorylation of Calponin and Caldesmon myosinATPase is no longer inhibited, resulting in increased activity; similarto the observations described herein.

Thus the spasminogen may be a molecular entity which is itself aphosphatase inhibitor or an activator of a protein kinase. Alternativelyit may be a molecular entity which switches on a phosphatase inhibitoror which stimulates the production of a phosphatase inhibitor. It isalso possible that the spasminogenic effect is brought about by a groupof compounds which in concert, act to inhibit a phosphatase or activatea protein kinase. Thus the molecular entity which brings aboutinhibition of phosphatase may be acid stable, with hydrophobic andhydrophilic domains and a mw of less than 1000 daltons. Thus theinventors provide that the spasminogen is a molecular entity (or groupof compounds) which brings about phosphatase inhibition or activation ofa protein kinase. The molecular entity which switches on a phosphataseinhibitor or which stimulates the production of a phosphatase inhibitor.The spasminogen may comprise a group of compounds which in concert, actto inhibit a phosphatase. The molecular entity or one or more of saidgroup of compounds may be acid stable, with hydrophobic and hydrophilicdomains and a mw of less than 1000 daltons. The molecular entity or oneor more of said group of compounds may be an okadaic acid-like compound.Thus there is also provided a further new way of diagnosing patientslikely to suffer a pathological condition which is in consequence ofspasm of smooth muscle cells (such as chronic cerebral vasospasmfollowing from subarachnoid haemorrhage) which comprises obtaining atissue or fluid sample from a patient and analysing that sample for aninhibitor of a phosphatase or an activator of a protein kinase. Theanalysis may be for a phosphatase inhibitor which has one or more of thefeatures following: (a) acid stability; (b) mw of less than 1000daltons; (c) hydrophilic and hydrophobic domains; (d) lipophilic domain;(e) protein philic domain. The analysis may be for a phosphataseinhibitor which inhibits the de-phosphorylation of myosin ATPase. Theanalysis may be for a compound related to/similar to okadaic acid. Onemay analyse for an inhibitor of a phosphatase in accordance withtechniques which are known and available in the art.

The OxBox and methods as described earlier which measure a marker ofoxidative metabolism are sensitive, reliable and quantitative Thereforethey may be used as a more general test to aid in diagnosis of andscreen for tests involving phosphatase inhibition.

Further provided is the use of a phosphatase inhibitor or protein kinaseactivator (eg as set out above) to screen for compounds useful as drugsto counter or block their spasminogenic effects on smooth muscle. Forexample in the screening method described earlier herein, thespasminogenic compound of the incubation may comprise a phosphataseinhibitor such as okadaic acid.

Further provided are treatment methods and medicaments for conditionsassociated with chronic spasm constriction eg vasospasm/vasoconstrictionwhich are based on the use of a compound which blocks the inhibitoryeffects of the spasminogen/spasminogenic group of compounds. Thus thetreatment compound may block the inhibitory effects of a phosphataseinhibitor, or it may block the switching on of a phosphatase inhibitor,or the production of a phosphatase inhibitor. Such a compound maycomprise a specific binding partner for the phosphatase inhibitor. Wherethis is so, the specific binding partner would in essence be“mopping-up” the inhibitor, thereby reducing or preventing its normalinteraction with the phosphatase.

In relation to the above, a specific binding partner comprises a memberof a specific binding pair which have particular specificity for oneanother and which in normal conditions bind to each other in preferenceto other molecules. Examples of specific binding pairs are antigens andantibodies (eg in the example stated above the phosphatase inhibitorwould be the antigen in the context of an immuno-based specific bindingpair); ligands and receptors; ligands and enzymes; enzymes andsubstrates; and complementary nucleotide sequences. The skilled personwill be able to think of many other examples and there is no need tolist them all here. Further the term “specific binding pair” is alsoapplicable where either or both of the constitutive elements comprisepart of a larger molecule.

Alternatively, the compound may comprise a different non-functional (inthe sense of not inhibiting the normal catalytic action of thephosphatase) ligand for the phosphatase ie a mimic (“mimetic”) of theinhibitor. Where this is so, the phosphatase binding sites available forinteraction with the inhibitor are effectively reduced, thereby leadingto a reduction in the inhibitory effects of the ligand.

The designing of mimetics and structural analogues (such as antagonisticstructural analogues) is a known approach to the development ofpharmaceuticals based on a lead compound.

In order that the present invention is clearly understood, embodimentsand examples will now be described by way of illustration only withreference to the figures referred to below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simple diagrammatic representation of the measurement device(hereafter called a “OxBox”).

FIG. 2 shows the effect of blood or sub-arachnoid CSF supernatant from apatient with chronic cerebral vasospasm on oxygen consumption(micromoles/min/g dry weight) of porcine carotid artery over a period of5 hours; A=sample of sub-arachnoid haemorrhage supernatant; B=peripheralblood sample; C=control sample.

FIG. 3 shows the effect of blood or sub-arachnoid CSF supernatant from apatient with chronic cerebral vasospasm on oxygen consumption(micromoles/min/g dry weight) of rat aorta over a period of 4.5 hours;A=sample of sub-arachnoid haemorrhage supernatant; B=peripheral bloodsample.

FIG. 4 compares the effect of blood from a patient with chronic cerebralvasospasm (B) with blood from a patient not having chronic cerebralvasospasm (C) on oxygen consumption (micromoles/min/g dry weight) ofporcine carotid artery over a period of 7 hours.

FIG. 5 shows the effect of sub-dual haemorrhage supernatant on oxygenconsumption (micromoles/min/g dry weight) or porcine carotid artery overa period of 5 hours; C=control sample; D=supernatant from the sub-duralspace.

FIG. 6 shows the effect of intraventricular haemorrhage supernatant onoxygen consumption (micromoles/min/g dry weight) of porcine carotidartery over a period of 5 hours; C=control sample; E=intraventricularhaemorrhage supernatant.

FIG. 7 shows the effect of histamine and sub-arachnoid haemorrhagesupernatant on the oxygen consumption (micromoles/min/g dry weight) ofhuman basilar artery over a period of 3 hours; A=sample of sub-arachnoidhaemorrhage supernatant; F=pretreatment with histamine; C=controlsample.

FIG. 8 shows the effect of okadaic acid on oxygen consumption(micromoles/min/g dry weight) of porcine carotid artery over a period ofa period of about 5 hours.

FIG. 9 shows the closely comparable effects of okadaic acid andsub-arachnoid CSF supernatant from a patient with chronic cerebralvasospasm on oxygen consumption (micromoles/min/g dry weight) over aperiod of about 5 hours.

FIG. 10 shows rate of oxygen consumption (micromoles/min/g dry weight)by porcine carotid arteries in the presence of blood plasma frompre-eclamptic, normal pregnant and non-pregnant women and in the absenceof plasma.

FIG. 11 shown individual rates of oxygen consumption (micromoles/min/gdry weight) at 90 minutes by porcine carotid arteries in the presence ofblood sera from pre-eclampsic patients and their matched pregnantcontrols.

FIG. 12 shows the shows rate of oxygen consumption (micromoles/min/g dryweight) by porcine carotid arteries in the presence of CSF from patientswith Alzheimer's disease.

FIG. 13 is a representation of the steady state rate of oxygenconsumption (micromoles/min/g dry weight) by porcine carotid arteries inthe presence of CSF from patients with Alzheimer's disease and controlpatients.

FIG. 14 shows actual oxygen consumption (micromoles/min/g dry weight) byporcine carotid arteries in the presence of CSF from individualAlzheimer's disease and control patients. The * marks the point at whichin a test with CSF from an Alzheimer Disease patient, the CSF is rinsedaway.

The examples and embodiments described herein serve to illustrate thepresent invention and are not to be interpreted as limiting its scope.

Oxygen consumption of a tissue or cell sample can be measured by use ofa simple device as represented in FIG. 1 and which the inventors call anOxBox.

A sample of tissue comprising muscle cells such as porcine carotidartery (14) is dissected out at 4° C. from source eg a recently killedpig and the surrounding tissues removed. The tissue sample is maintainedat 30° C. in an airtight vessel (10) in a previously aerated (18)solution (12) for supporting oxidative metabolism. The vessel (10)should be heat and light resistant and it may be made of any materialproviding such qualities eg glass, perspex, plastic. Other materialswill be known to those skilled in the art.

Any tissue appropriate to the type of vasospasm under study may be used.For example if one is concerned with the diagnosis of cerebralvasospasm, porcine carotid tissue or basilar artery tissue may be used.If one is diagnosing cardiac vasospasm, suitable coronary artery tissuemay be selected. Alternatively, one may use a culture of suitable musclecells. Species and vascular bed cross-over is perfectly possible andexemplified.

A suitable solution (12) for maintenance of the tissue sample andoxidative metabolism is Krebs buffer with 11 mM glucose and/or 5 mMpyruvate. An alternative solution is Hepes. Others appropriate to themaintenance of the tissue or cells being used will be known to thoseskilled in the art. The pH should be around 7.4.

An oxygen sensor (16) is suspended in the solution for the measurementof oxygen consumption over suitable period of time (eg 1 to 5 hours)under varied experimental conditions.

EXAMPLE 1

An approximately 0.5 g sample of porcine carotid artery is used in anOxBox as generally described above. The sample is immersed in 6 ml ofbuffer solution, exposed to air (approx. 20% oxygen) with a restingoxygen consumption rate of 0.2 μMol/mg wet weight tissue/min. The tissuesample is maintained in these conditions for 30 mins, and the oxygenconcentration in the chamber measured over a period of time to give thetissue's basal rate of oxygen consumption in non-working conditions.

A 200 μls sample of either sub-arachnoid haemorrhage CSF supernatant (A)or peripheral blood serum (B) (blood collected in a collection tubecomprising a suitable coagulant such a sodium citrate; the serumproduced by spinning down the blood or supernatant at 1000 rpm for 20min.; it its preferable to avoid use of heparin as the anticoagulant asit can upset intracellular signalling mechanisms) from a patient inchronic cerebral vasospasm is added to the vessel and the oxygenconcentration measured over a period of 300 minutes in order todetermine any change in the rate of oxygen consumption.

Suitable controls (C) are set up. These controls may simply be withoutsample. Alternatively, they may comprise biological samples known not tocontain a spasminogen eg human serum or CSF from a patient withhydrocephalus, but not in vasospasm. Otherwise identical experimentalconditions are used.

As can be seen from FIG. 2, a 5x increase in the rate of oxygenconsumption is observed for sub-arachnoid haemorrhage supernatant (A).This comprises a first demonstration of a vasoactive compound in CSF.Peripheral blood (B) from such a patient, although causing a definiteand consistent increase in the rate of oxygen consumption, does notproduce an effect as marked as sub-arachnoid haemorrhage supernatant.Controls (C) as discussed above, do not have an effect on oxygenconsumption.

EXAMPLE 2

The experiment is conducted as for Example 1, except that rat aorta isused instead of porcine carotid artery. The test samples are (A) and (B)as above. The results are shown in FIG. 3. As in Example 1 above, a 5xincrease in the rate of oxygen consumption is observed for sub-arachnoidhaemorrhage supernatant (A) and the increase in oxygen consumptionassociated with peripheral blood (B) is not so marked. The experimentshows that the results shown in FIG. 2 are not specific to porcinecarotid artery.

EXAMPLE 3

The experiment is conducted as for Example 1, except that this timeperipheral blood from a sub-arachnoid haemorrhage patient with chroniccerebral vasospasm (B) is compared with peripheral blood from a patientnot in chronic cerebral vasospasm (C).

As can be seen from FIG. 4, peripheral blood from a patient in chronicvasospasm causes about a 2x increase in oxygen consumption, whereasperipheral blood from the control patient showed only a very smallincrease of no significance. In order to better observe the effects onoxygen consumption caused by peripheral blood from a patient in chroniccerebral vasospasm, it is advantageous to increase the incubation time.This can be seen from a comparison of FIGS. 2 and 4.

EXAMPLE 4

The experiment is conducted as for Example 1, except that this time thetest samples comprise either supernatant haemorrhage (E) of a patientwith chronic cerebral vasospasm following intraventricular haemorrhage.As can be seen from FIG. 5, supernatant from the sub-dural space doesnot increase oxygen consumption. FIG. 6 shows that intraventricularhaemorrhage supernatant although causing an increase in oxygenconsumption, has an attenuated effect in that it takes about 3.5 hoursfor the increase in oxygen consumption to start. As can be seen fromFIG. 2, supernatant from the sub-arachnoid space of a patient in chroniccerebral vasospasm, causes an almost immediate increase in the rate ofoxygen consumption. This result suggest that in sub-arachnoidhaemorrhage vasospasm the spasminogen may be associated with the mixingof blood and CSF of the sub-arachnoid space.

EXAMPLE 5

The experiment is conducted as for Example 1, except that human basilarartery is used instead of porcine carotid artery. The experimentinvestigates the effects of histamine pre-treatment. If the artery ispre-treated with histamine (F) (histamine concentration of 10⁻⁴ molar inthe 6 mls of OxBox buffer for 1 hour) the large increase in oxygenconsumption in response to supernatant from the sub-arachnoid space of apatient in chronic vasospasm (a) was abolished. The effect is shown inFIG. 7.

EXAMPLE 6

Porcine carotid arteries are maintained in ice cold physiological saliesolution (PSS) containing (in mM) 116 NaCl, 25 NaHC03, 5.4 KCL, 5KH2PO4, 1.2 CaCl2, 1.25 MgSO4 and 11 glucose. The arteries are debridedof fat and connective tissue at 4° C. 2-3 g wet weight of arteries areplaced in a 10 mm diameter NMR tube and superfused at 37° C. at aconstant flow rate of 20 ml/min with Krebs-Hensleit buffer containing0.5 mM KH2PO4 with either 11 mM glucose or 5 mM pyruvate as thesubstrate and aerated with 95%O2−5%CO2. The arteries were perfused for90 min in control media and then in the presence of Krebs-Hensleitbuffer supplemented with 1 part to 30 sub-arachnoid haemorrhagesupernatant collected from four patients with angiographic vasospasm.The carotids are equilibrated for 20 minutes in the NMR tube and theATPase flux measured in a 400 MHz magnet interfaced to a Brukerspectrometer.

ATPase flux (ATP to ADP) increases 6 fold (0.02-0.12 μmol/g/s) and issustained for 18 hrs. Adenylate kinase (Pi+ADP to ATP) fails to keeppace with ATPase flux and therefore ADP levels increase (41 to 114μmol/L).

Therefore haemorrhagic CSF caused a large and sustained increase inATPase activity; far more than any known spasminogens orvasoconstrictors. The haemorrhagic CSF appears to directly stimulateactinomysin ATPase in smooth muscle. The mitochondria respond, but areunable to maintain metabolic homeostasis and ADP increases.

EXAMPLE 7

The OxBox as described herein can be used to monitor the treatment ofchronic cerebral vasospasm or other such conditions such as cardiacvasospasm. The present example concerns monitoring treatment of chroniccerebral vasospasm with dobutamine. 1 day following the onset ofsub-arachnoid haemorrhage, a blood or CSF sample from a patient isdiagnosed as being able to stimulate eg porcine carotid artery toincrease oxygen consumption and therefore as comprising a spasminogen(see Example 1) Such a sample is described as being “hot”. The patientis started on a suitable vasoactive agent such as dobutamine eg 500 mgdobutamine in 50 mls normal saline. Infusion commencing at 2.5 μg/kgbody weight/min. rising to 10 μg/kg body weight/min. Infusion may becontinued for up to 10 days. (Dobutamine is conventionally used in thetreatment of renal failure and high blood pressure). The patient ismonitored by use of the Ox-Box on eg days 2, 4 and 8, until the samplewithdrawn from the patient turns “cold” ie no longer able to stimulateporcine carotid artery to increase oxygen consumption.

Typically the sample will turn cold within hours of dobutamine infusionindicating the effectiveness of treatment with dobutamine.

EXAMPLE 8

The OxBox as described herein can be used to screen for new drugs totreat conditions such as chronic cerebral vasospasm which result formspasm of smooth muscle cells. As shown in Example 5 pretreatment ofhuman basilar artery tissue with histamine reduces the rate of oxygenconsumption to near that of the control upon application ofsub-arachnoid haemorrhage supernatant (FIG. 7), when measured over aperiod of 180 minutes. Histamine is known to put vessels into acontracted state and the present inventors believe that this contractedstate may protect the stimulation of ATPase caused by the supernatant.

Thus the OxBox and method as described herein can be used to look fordrugs which block the spasminogenic effects of the CSF. The resultsshown in FIG. 7 indicate the effectiveness of histamine as a treatmentto prevent chronic cerebral vasospasm.

EXAMPLE 9

The OxBox as described herein can be employed in the elucidation ofspasminogens. Suspected spasminogens can be tested for increasing oxygenconsumption of eg porcine carotid artery as described in Example 1. Inone such test, porcine carotid artery was stimulated with 1 nm okadaicacid and the oxygen consumption rate measured over a period of about 5hours. The results are shown in FIGS. 8 and 9. It can be seen thatokadaic acid causes an increase in oxygen consumption strikingly similarto that caused by sub-arachnoid CSF supernatant from a patient withchronic cerebral vasospasm.

EXAMPLE 10

The experiment is conducted as for Example 1, except that this time theeffects of sera obtained from pre-eclampsic patients is compared withsera taken from normal pregnant and non-pregnant controls. The data (seeFIGS. 10 and 11) shows that metabolic changes in vessels exposed to serafrom pre-eclampsia patients are comparable to the metabolic changes invessels exposed to eg CSF from patients with sub-arachnoid haemorrhagevasospasm or okadaic acid an inhibitor of phosphatase type 2A.

In pre-eclampsia patients, there was a significant stimulation ofrespiration after 90 minutes when the porcine carotid arteries wereexposed to the serum of pre-eclampsia patients (when compared to matchedcontrols; FIG. 10; n=17 matched pairs). Frequently a five fold increasein oxygen consumption was seen in resting (non-contracting) porcinecarotid artery in response to serum from pre-eclampsia patients.

Moreover, in a comparison of pre-eclampsic and pregnant control serathere is a striking acceleration of respiration, which is significantlygreater than control FIG. 11). All experiments were performed at 30° C.on resting, non-contracting vessels.

Interestingly, the spasminogens causing cerebral vasospasm stimulatingvessels during pre-eclampsia may not be the same. Two differences pointto this. Firstly, the pre-eclampsia stimulation is completely reversibleby eg rinsing, whilst cerebral vasospasm stimulation is not. Secondly,the stimulation could be prevented if the cerebral vasospasm CSF waspre-treated with esterase. In contrast esterase treatment had no effecton pre-eclampsic sera's stimulation of the vessels.

EXAMPLE 11

The experiment is conducted as for Example 1, except that this time theeffects of CSF obtained from Alzheimer's Disease patients is comparedwith CSF from individuals without the disease. The data (see FIGS. 12,13 and 14) shows that metabolic changes in vessels exposed to CSF fromAlzheimer's Disease patients are comparable to the metabolic changes invessels exposed to eg CSF from patients with sub-arachnoid haemorrhagevasospasm or okadaic acid an inhibitor of phosphatase type 2A/

There was a significant stimulation of respiration after 60 minutes.FIG. 12 shows the rate of oxygen consumption for porcine carotidarteries contacted with CSF from patients with clinically diagnosedAlzheimer's disease (G) and patients without Alzheimer's disease (H).CSF was taken from both groups at the time of post-mortem examination.CSF was added to the standard respiration solution bathing a sample ofporcine carotid artery (see earlier). As in Example 1, 1 part (eg 200μls) CSF was added to 30 parts (6 mls) of respiration solution and therate of respiration measured for at least 90 minutes. As can be seenfrom FIG. 12, CSF from Alzheimer's disease is associated with animmediate and significant increase in respiration. CSF from controlpatients had no such effect. Error bars are standard deviation.

FIG. 13 is a graphical representation of the steady state rate of oxygenconsumption (ie the rate plotted are those taken at the time when therate of oxygen consumption had plateaued) in an OxBox system for CSFfrom Alzheimer's Disease and control patients. As can be seen, CSF frommost Alzheimer's Disease patients caused a steady state respiration ratesignificantly higher than caused by CSF from normal patients.

FIG. 14 shows the actual respiration rates for porcine carotid arteriesexposed to CSF from each individual Alzheimer's Disease and controlpatient. The * marks the point at which in a test with CSF from anAlzheimer disease patient, the CSF was rinsed away. The removal of CSFfrom an Alzheimer's Disease patient caused the respiration rate torapidly fall, reaching about the rate of CSF from control patientswithin about 30 mins. This effect of removal of Alzheimer's Disease CSFon respiration rate is similar to that observed using serum frompre-eclampsia patients and distinct to the effect observed using CSF orserum from sub-arachnoid haemorrhage patients.

The above provides demonstration of a vasoactive agent in CSF ofAlzheimer's Disease patients. There has been no previous suggestion of avasoactive eg vasoconstrictive agent being involved in Alzheimer'sDisease.

The fact that the effects caused by CSf from Alzheimer's diseasepatients are reversible, indicates that the spasminogen(s) concerned canbe dissociated from its receptor or binding site. The OxBox system alsoallows for the identification and manufacture of molecular entitieswhich are structurally related to the spasminogen (eg analogues thereof)which although able to bind to the receptor/binding site for thespasminogen do not stimulate the pathological effects. Any suchmolecular entities are likely to be of practical value as drugs.

A screen for such molecular entities eg analogues may be carried outwith or without specific knowledge of the nature of the spasminogen fora pathological effect. Various screening approaches may be employed. Forexample, the OxBox system as described herein can be set up using CSFfrom an Alzheimer's disease patient to establish a positive respiratoryresponse ie a significant rise in the rate of oxygen consumption. Onecan then add a molecular entity to the respiration solution and observethe rate of oxygen consumption. Since the spasminogen(s) associated withAlzheimer's disease can be dissociated from its receptor/binding sitewith a consequent drop in the rate of oxygen consumption, a drop in therate of oxygen consumption caused by the addition of the molecularentity to the respiration solution, indicates that the molecular entityis able to compete with the spasminogen for binding to the spasminogenreceptor/binding site.

Molecular entities with such effects are likely to be of value astreatment (prophylactic/therapeutic) agents.

One suitable screening approach is described above. Others will beapparent to those skilled in the art.

Such a screen as set out may be used to look for treatment agents inrelation to other pathologies associated with spasminogenic agents egpre-eclampsia.

A molecular entity identifies as being able to compete with aspasminogen for binding to the spasminogen receptor/binding site may bepolypeptide, peptide or non-peptide in nature. Non-peptide “smallmolecules” are often preferred for many in vitro pharmaceutical uses.Accordingly, a mimetic or mimic of the substance (particularly if apeptide) may be designed for pharmaceutical use.

The designing of mimetics and structural analogues (such as antagonisticstructural analogues) to a known pharmaceutically active compound is aknown approach to the development of pharmaceuticals based on a “lead”compound (eg the molecular entity identified as being of interest by theOxBox system). This might be desirable where the active compound isdifficult or expensive to synthesise or where it is unsuitable for aparticular method of administration, e.g. peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large number of moleculesfor a target property.

There are several steps commonly taken in the design of a mimetic orstructural analogue (such as an antagonistic structural analogue) from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,eg by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmoacophore”.

Once the pharmacophore has been found, its structure is modelled toaccording its physical properties, eg stereochemistry, bonding, sizeand/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic or structural analogue.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide based, further stability can be achieved by cyclising thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Given the above, the present invention also provides molecular entitiesand treatment agents as set out above and readily and routinelyavailable by the approaches as set out above, pharmaceuticals comprisingsuch molecular entitites/treatment agents, uses of such molecularentities/treatment agents eg in screening systems, design of mimeticsand structural analogues, formulation and use of pharmaceuticals.

What is claimed is:
 1. A method for screening compounds for potentialefficacy as therapeutic agents which comprises incubating, in a mediumsuitable for the support of oxidative metabolism, a cell or tissuesample which comprises smooth muscle cells with (a) a specimen, agent orcombination of agents known to effect an increase in oxidativemetabolism of the cell or tissue sample which is not attributable to acontractile demand for ATP; and (b) a test compound; measuring a markerof oxidative metabolism; and detecting an absence or reduction of theincrease in oxidative metabolism not attributable to a contractiledemand for ATP and which is normally effected by the specimen, agent orcombination of agents, any such absence or reduction being indicative ofthe test compound having potential efficacy as a therapeutic agent.
 2. Amethod according to claim 1 wherein the cell or tissue sample comprisesvascular smooth muscle cells.
 3. A method in accordance with claim 1wherein identification of a test compound having potential efficacy as atherapeutic agent includes preparation of a synthetic version orderivative of the test compound which substantially retains thebiological activity of the test compound.
 4. A method in accordance withclaim 1 or 3 wherein identification of a test compound having potentialefficacy as a therapeutic agent includes preparation of a syntheticversion or derivative of the test compound, and wherein a pharmaceuticalcomprising said test compound or derivative in a therapeuticallyeffective amount is prepared.
 5. A method according to claim 1 whereinthe specimen is selected from cerebral spinal fluid (CSF); blood, seraor plasma.
 6. A method according to claim 1 wherein the marker ofoxidative metabolism is selected from oxygen consumption rate, ATPaseactivity, kinase activity phosphorylation state of myosin or ADPconcentration in the cell or tissue sample.
 7. A method according toclaim 1 wherein said medium comprises Krebs buffer with 11 mM glucoseand/or 5 mM pyruvate or Krebs-Hensleit buffer containing 0.5 mM KH₂PO₄with 11 mM glucose and/or 5 mM pyruvate.
 8. A method according to claim1 wherein said medium is free of viable microorganisms.
 9. A methodaccording to claim 1 wherein the incubation is aerated with 95% O₂ and5% CO₂.
 10. A method according to claim 1 wherein the incubating isconducted at a pH which supports oxidative metabolism of the cell ortissue sample.
 11. A method according to claim 1 wherein the incubatingis conducted a pH of at substantially 7.4.